Separations of biological objects, such as proteins, chromosomes, nucleic acids, cells, organelles, and the like, and other types of objects are important in various detection, isolation, quantification, and diagnostic processes. Specificity and sensitivity are two important parameters that are generally desired in these separation schemes.
Hybridization probes are widely used to detect and/or quantify the presence of a particular nucleotide sequence in a mixed sample of nucleotide sequences. Hybridization probes detect the presence of a particular nucleotide sequence, referred to herein as a target sequence, through the use of a complementary nucleotide sequence that selectively hybridizes to the target nucleotide sequence. For a hybridization probe to hybridize to a target sequence, the hybridization probe must contain a nucleotide sequence that is complementary to the target sequence. The complementary sequence must also be sufficiently long for the probe to exhibit selectivity for the target sequence over non-target sequences.
Hybridization assays can be designed to detect the presence or absence of a particular nucleotide sequence, for example the presence of a gene in a DNA sequence. Hybridization assays can also be designed to detect the movement of a nucleotide sequence relative to another nucleotide sequence in a sample, for example the presence of a gene on a chromosome that is known to be normally located on a different chromosome, e.g., the detection of the abl gene on chromosome 22 in human leukemia patients (e.g., Tkachuk et al., 250 Science 559–562 (1990); C-TRAK translocation detection system commercially available from Oncor, Inc., Gaithersburg, Md.; U.S. Pat. No. 5,447,841; U.S. Pat. No. 5,731,153; and U.S. Pat. No. 5,783,387).
As used herein, “nucleotide sequence aberrations” refers to rearrangements between and within nucleic acids, particularly chromosomal rearrangements. “Nucleotide sequence aberrations” also refers to the deletion of a nucleotide sequence, particularly chromosome deletions. As used herein, the term “nucleic acids” refers to both DNA and RNA.
A chromosome translocation is an example of a nucleotide sequence aberration. A chromosome translocation refers to the movement of a portion of one chromosome to another chromosome (inter-chromosome rearrangement), as well as the movement of a portion of a chromosome to a different location on that chromosome (intra-chromosome rearrangement). In general, chromosome translocations are characterized by the presence of a DNA sequence on a particular chromosome that is known to be native to a different chromosome or different portion of the same chromosome. Because chromosome translocations involve the movement of a nucleotide sequence within a sample, as opposed to the appearance or disappearance of the nucleotide sequence, it generally is not possible to detect a chromosome translocation merely by assaying for the presence or absence of a particular nucleotide sequence.
Chromosome translocations are known to increase in frequency upon exposure to radiation and certain chemicals. Measurement of the frequency of chromosome translocations after exposure to radiation or a particular agent is therefore useful for evaluating the tendency of such agents to cause or increase the frequency of chromosome translocations. Also, the frequency (translocations per cell) of chromosome translocations measured in blood lymphocytes from an individual can be used as a quantitative measure of the amount of prior exposure to such agents (e.g., T. Straume and J. Lucas “Validation studies for monitoring of workers using molecular cytogenetics,” Biomarkers in Occupational Health: Progress and Perspectives (M. L. Mendelsohn, J. P. Peeters, and M. J. Normandt, Eds.), Joseph Henry Press, Washington D.C., pp. 174–193 (1995)).
Chromosome translocations are also known to be associated with specific diseases, including, for example lymphomas and leukemia, such as Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia, and granulocytic leukemia, as well as solid tumors such as malignant melanoma, prostate cancer, and cervical cancer. A method for efficiently detecting a translocation associated with a disease is needed as a method for diagnosing disease, follow-up of cancer therapy patients, research, and population studies.
Fluorescence in situ hybridization (FISH) using chromosome-specific composite hybridization probes (“chromosome painting”) was developed as an assay for detecting chromosome translocations. FISH and selected applications of the FISH method are described in Pinkel et al., 83 Proc. Nat'l Acad. Sci. USA 2934–2938 (1986); Straume et al., UCRL 93837 (1986); Pinkel et al., 85 Proc. Nat'l Acad. Sci. USA 9138–9142 (1988); U.S. Pat. No. 5,447,841; Lucas et al., 62 International Journal of Radiation Biology 53–63 (1992); Straume et al., 62 Health Physics 122–130 (1992); Straume and Lucas, 64 Int. J. Radiat. Biol. 185–187 (1993).
The fluorescent hybridization probes used in FISH-based chromosome painting are substantially chromosome-specific, i.e., they hybridize primarily to a particular chromosome type. Unique or substantially unique probes may be used to limit non-specific hybridization. A discussion of so-called unique, middle repetitive, and highly repetitive sequences and their implications for hybridization probes is found in U.S. Pat. No. 5,447,841. Chromosome translocations are identified in the FISH assay by visually scanning individual cells for the presence of two different fluorescent signals on a single chromosome, the two fluorescent signals originating from two different cocktails of FISH probes, each probe cocktail having homology to a different chromosome type.
Because each FISH probe hybridizes to a specific chromosome type and not to the chromosome translocation itself, it is not possible to determine the frequency of chromosome translocations directly from the fluorescence signal emanating from a FISH probe. Rather, the frequency of chromosome translocations in a cell sample must be determined according to FISH assays by visually scanning individual metaphase cells on slides and identifying whether the two fluorescent signals appear on the same chromosome. The need to visually scan such individual cells effectively limits the number of cells that can be assayed, thereby reducing the sensitivity of the FISH assay, introducing the possibility of human error, and greatly increasing cost per analysis.
Accordingly, a fast, accurate method is needed for quantifying chromosome translocations and other nucleotide sequence aberrations. In particular, a method is needed that can isolate and quantify nucleotide sequence aberrations contained in the nucleic acid of a sample of cells without the need to analyze each cell individually.
U.S. Pat. No. 5,731,153 relates to a two-step separation procedure that uses two solid supports, each coated with unique complexing agents that bind to hybridization probes complementary to different target sequences. This procedure requires detachment of the target sequence from the first solid support after the first separation step and reattachment of the target sequence to a second solid support before the second separation step can be performed. The requirement for re-attachment of the target sequence is particularly problematic and would add significantly to the complexity and cost of commercial separation kits using such methodology and reduce the precision of the assay because of variability in the detachment/reattachment step. Further, this procedure is limited to two types of solid supports, but it would be useful to have more support options to facilitate multiple simultaneous analyses. Moreover, the preferable methods for quantification described in U.S. Pat. No. 5,731,153 require either very expensive and uncommon equipment (e.g., measure 14C by accelerator mass spectrometry) or much less quantitative methods such as the detection of fluorescence labels on reporter nucleic acid probes. Also, the method in U.S. Pat. No. 5,731,153 is limited to separation of nucleic acids, whereas it would be advantageous to separate other types of objects as well.
Unfortunately, methods available for the quantification of chromosomal rearrangements are either very costly and inefficient, e.g., cytogenetic-type analyses (H. J. Evans et al., 35 Chromosoma 310–325 (1971); D. Pinkel et al., 83 Proc. Natl. Acad. Sci. USA 2934–2938 (1986); D. Pinkel et al., 85 Proc. Natl. Acad. Sci. USA 9138–9142 (1988); D. C. Tkachuk et al., 250 Science 559–562 (1990)), or require small sequences such as fusion mRNAs that may be amplified by PCR and detected (M. H. Delfau et al., 4 Leukemia 1–5 (1990); A. Zippelius & K. Pantel, 906 Annals NY Acad. Sci. 110–123 (2000)). Cytogenetics require a highly trained technician to visually score metaphase or interphase cells using a microscope and make judgements about what is observed. PCR is less labor intensive than cytogenetics but is of limited utility in direct DNA-based detection of most chromosomal translocations because the fusion points tend to be variable. D. C. Tkachuk et al., 250 Science 559–562 (1990); E. Solomon et al., 254 Science 1153–1160 (1991). These limitations have essentially restricted PCR to the detection of fusion mRNAs, which may not always be known, may arise from ectopic expression, or may be expressed deficiently (A. Zippelius & K. Pantel, 906 Annals NY Acad. Sci. 110–123 (2000)).
In view of the foregoing, it will be appreciated that providing a separation method that does not require reattachment of the target sequence to a solid support to perform the second step, does not require PCR, is highly quantitative, can be accomplished using readily available laboratory equipment, can be used for multiple simultaneous analyses, and that is applicable to the isolation and quantitation of many different kinds of objects, including nucleic acids, metaphase chromosomes, proteins, cells, organelles, and the like, would be a significant advancement in the art.
Such methods are disclosed herein.